Fig. 3. ALE2 is an active protein kinase. (A) In vitro kinase activity of ALE2. GST-fusion proteins of the putative protein kinase domain of ALE2 (GST:ALE2KD) were tested to determine whether they have autophosphorylation activities (upper panel). Kinase activity of GST:ALE2KD was determined with MBP as a substrate (lower panel). (B) In vitro kinase activity of ACR4. The His-Nus-fusion protein of the ACR4 protein kinase domain autophosphorylated and phosphorylated MBP in vitro. (C) A mutual phosphorylation between ALE2 and ACR4. Effects of ALE2 or ACR4 on the activity of the other kinase were determined by kinase assays in vitro using an equal amount of recombinant ALE2KD and ACR4KD proteins. Four possible combinations of wild-type and kinase-inactive mutant proteins were examined. (D) Quantification of phosphorylation activities. The amounts of [-³²P] ATP incorporated into the substrate proteins were determined. Wild-type ALE2KD and ACR4KD proteins increased the phosphorylation of kinase-inactive ACR4KD and ALE2KD, respectively (arrows). When either wild-type ALE2KD or ACR4KD protein was used as a substrate, a synergistic increase of phosphorylation by the other protein was observed (asterisks).